Culicoides obsoletus allergens for diagnosis of insect bite hypersensitivity in horses

AInsect Bite Hypersensitivity (IBH) is the most common skin allergy in horses and involves a Type I (IgE mediated) hypersensitivity reaction against bites of insects, mainly of the Culicoides species. Welfare of affected horses is seriously reduced and no fully curative treatment is yet available. Furthermore, current diagnostic tests are unreliable because of their low sensitivity and specificity. Aim of our research was to increase the understanding of immunological aspects of IBH, with special attention to improving diagnosis by the characterization and production of recombinant allergens. Whole body extracts (WBE) of three Culicoides species: C. obsoletus C. nubeculosus and C. sonorensis were evaluated for their applicability for diagnosis of IBH in horses in The Netherlands. They were tested for IgE binding by ELISA and Western blotting and for their capacity to degranulate basophils in a histamine release test. For all tests, best results were obtained with C. obsoletus. The ELISA was further evaluated using C. obsoletus extract on approximately 200 IBH affected and healthy horses, which demonstrated high test sensitivity and specificity. C. obsoletus-specific IgE serum levels were found to be the same in the IBH season and off season, suggesting that the test can be used to diagnose horses in winter when clincial symptoms are absent. Since C. obsoletus was found to be the most important species for diagnosis of IBH in The Netherlands, mRNA of this Culicoides species was sequenced and assembled to create a transcriptome. Using the sequences from in literature described allergens from C. nubeculosus and C. sonorensis, similarity searches were performed on this transcriptome,. This resulted in the identification of seven allergens from C. obsoletus. These allergens were cloned and expressed as recombinant proteins in E. coli and named Cul o 1 – Cul o 7. The frequency of positive test results by ELISA within IBH affected horses ranged from 38 % to 67 %. The capability of the allergens to induce Type I hypersensitivity reaction in IBH affected horses was demonstrated by an intradermal test. The applicability of the 7 C. obsoletus derived recombinant allergens was further evaluated and compared with C. obsoletus WBE in an IgE ELISA using a large number of horses.The highest test accuracy was obtained with WBE, followed by Cul o 2, 3 and 5. Two ELISA’s with a combination of recombinant allergens, combi-1 (Cul o 3, 5 and 7) and combi-2 (Cul o 1, 2, 5 and 7) were additionally performed and both resulted in high test accuracies close to that obtained with WBE. Both combi-1 and combi-2 resulted in a lower test sensitivity with samples collected in winter compared to samples collected in IBH season, but most IBH affected horses could still also be correctly diagnosed in winter. The association between several factors and IgE levels against C. obsoletus whole body extract and the 7 recombinant allergens was quantified. Furthermore, the relation between IgE levels and severity of symptoms was examined. Severity of symptoms and IgE levels against several C. obsoletus allergens were found to be related. Factors that were found to be associated with IgE levels were: breed, age, month of scoring, interaction between IBH status and month of scoring, degree of itchiness and number of seasons horses were affected with IBH. The general discussion discussed the prospects to use the produced recombinant allergens for immunotherapy treatment of IBH affected horses. The panel of all 7 recombinant allergens allows to determine for which exact components of C. obsoletus the IBH horses are allergic (“component resolved diagnosis”). This will enable a tailor made composition of (recombinant) allergens for use in immunotherapy.</p

“Always looking for a new balance”: toward an understanding of what it takes to continue working while being diagnosed with relapsing-remitting multiple sclerosis

Background: The aim of this study was to gain insight into the meaning of work in the everyday lives of people with relapsing-remitting multiple sclerosis, and the barriers and facilitators to staying in work.Methods: Nineteen employed adults diagnosed with relapsing-remitting multiple sclerosis participated in narrative interviews. All interviews were transcribed and coded for thematic analysis.Results: For people with relapsing-remitting multiple sclerosis, continuing to work was a precarious balancing act. Five themes influenced this balance: becoming familiar with the disease, adjusting expectations, having an understanding and realistic line manager, seeing work as meaningful life activity and strategic considerations.Conclusions: People receiving a diagnosis of relapsing-remitting multiple sclerosis have to refamiliarize themselves with their own body in a meaningful way to be able to continue their work. Rehabilitation professionals can support them herein by taking into account not merely functional capabilities but also identity aspects of the body. Medication that stabilizes symptoms supports making the necessary adjustments. A trusting relationship with the line manager is vital for this adaptation process. Additionally, a match between being adequately challenged by work, while still having the capacity to meet those work demands, is needed, as is long-term financial stability.Implications for rehabilitationRehabilitation professionals can support employees with relapsing-remitting multiple sclerosis by taking into account not merely functional capabilities but also identity aspects of the body.A trusting relationship with the line manager, including a timely disclosure of the diagnosis, is vital for people with relapsing-remitting multiple sclerosis to remain at work.For people with relapsing-remitting multiple sclerosis, there is a delicate balance between being adequately challenged by work while still having the capacity to meet work demands.FSW - Self-regulation models for health behavior and psychopathology - ou

tuf Gene dosage effects on the intracellular concentration of EF-TuB

In this paper we have studied the effect of raising the intracellular EF‐Tu concentration on the expression of tufB. To this aim cells were transformed with multicopy plasmids carrying either tufA or tufB. The intracellular EF‐Tu concentrations were determined by the specific immunoelectrophoresis assay described in the preceding paper in this journal. We have cloned the tufA gene in a plasmid, containing the powerful major leftward promoter (Pl) of phage λ Transcription from Pl can be repressed at low temperature by a temperature‐sensitive repressor and acitvated by heat induction. Cloning occurred in two orientations in a single EcoRI site about 150 base pairs downstream of Pl. Cells carrying either plasmid were shown to contain an almost doubled amount of EF‐Tu at temperatures from 28°C to 37°C. This indicates that transcription of tufA can proceed from a possible binding site for RNA polymerase on these cloned fragments. The EF‐Tu level was further increased to about 30% of total cellular protein after a temperature shift from 37°C to 43°C. The multicopy plasmid pTuB1 described by Miyajimaet al. [FEBS Lett. 102, 207–210 (1979)] and a derivative (pTuBo, compare preceding paper in this journal) were used to study the expression of both chromosomal and plasmid‐borne tufB. Transformation with either plasmid raised the intracellular EF‐Tu concentration by 30–60% depending on the nutritional conditions. Suppression of tufB expression was observed when the intracellular level of EF‐Tu increased after transformation with all plasmids mentioned above. The results are in accord with the concept that EF‐Tu acts as an autogenous feedback inhibitor involved in the regulation of tufB.Microbial Biotechnolog

The role of EF-Tu in the expression of tufA and tufB genes

Microbial Biotechnolog

A 38-year-old woman with necrotising cervical lymphadenitis due to Histoplasma capsulatum

Immunogenetics and cellular immunology of bacterial infectious disease